Key Product Details

Species Reactivity

Goat

Applications

Fluorophore-linked immunosorbent assay, Immunocytochemistry/ Immunofluorescence, Western Blot

Label

DyLight 649 (Excitation = 654 nm, Emission = 673 nm)

Antibody Source

Polyclonal Donkey IgG

Format

Pre-adsorbed

Concentration

LYOPH mg/ml

View all IgG (H+L) Secondary Antibodies »

Product Specifications

Immunogen

Goat IgG whole molecule

Specificity

This antibody was pre-adsorbed against Chicken, Guinea Pig, Hamster, Horse, Mouse, Rabbit, and Rat Serum Proteins. No reaction was observed against Chicken, Guinea Pig, Hamster, Horse, Mouse, Rabbit and Rat Serum Proteins. This antibody will react with heavy chains of Goat IgG and with light chains of most Goat immunoglobulins.

Clonality

Polyclonal

Host

Donkey

Isotype

IgG

Description

Store vial at 4C prior to restoration. For extended storage aliquot contents and freeze at -20C or below. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4C as an undiluted liquid. Dilute only prior to immediate use.

This product was prepared from monospecific antiserum by immunoaffinity chromatography using Goat IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Donkey Serum, Goat IgG and Goat Serum

Scientific Data Images

Western Blot: Donkey anti-Goat IgG (H+L) Secondary Antibody [DyLight 649] (Pre-adsorbed) [NBP1-72847] - Rabbit anti-Transferrin, Goat-anti-Alpha-1-Anti-Trypsin, and Mouse-a-GST. Detection shown was using DyLight549 Donkey anti-Rabbit IgG, DyLight 488 Donkey anti-Mouse IgG, and DyLight 649 Donkey anti-Goat IgG at 1:10000 (in blocking buffer min RT). Blots were washed, rinsed in methanol and dried.
Donkey anti-Goat IgG (H+L) Secondary Antibody [DyLight 649] (Pre-adsorbed)

Donkey anti-Goat IgG (H+L) Secondary Antibody [DyLight 649] (Pre-adsorbed)

Rabbit anti-Transferrin (109-4134 lot 3033, green), Goat-anti-Alpha-1-Anti-Trypsin ( lot 5842), and Mouse-a-GST ( lot 24882) were used in a multiplex system to detect target proteins under reducing (R) conditions (+4% BME) in albumin depleted human serum with 320 ng of added GST. Sample was run by SDS-PAGE, transferred to 0.2 um PVDF using the BioRad Trans-Blot Turbo and blocked in 2.5% Blotto, 2.5% BSA, 0.02% Tween over night at 4C. Membrane was probed with three primary antibodies at 1:1000 dilution (in over night at 4C). Detection shown was using DyLight549 Donkey anti-Rabbit IgG ( lot 21100, shown as green) DyLight 488 Donkey anti-Mouse IgG ( lot 21095, shown as blue), and DyLight 649 Donkey anti-Goat IgG ( lot 20834, shown as red) at 1:10000 (in 30 min RT). Blots were washed, rinsed in methanol, dried and Images were collected using the BioRad VersaDoc System.
Donkey anti-Goat IgG (H+L) Secondary Antibody [DyLight 649] (Pre-adsorbed)

Donkey anti-Goat IgG (H+L) Secondary Antibody [DyLight 649] (Pre-adsorbed)

DyLight(TM) dyes can be used for two-color Western Blot detection with low background and high signal. Anti-tubulin was detected using a DyLight(TM) 549 conjugate. Anti-TNFa was detected using a DyLight(TM) 649 conjugate. The image was captured using the Typhoon(TM) 9410 Imaging System.

Applications

Application
Recommended Usage

Fluorophore-linked immunosorbent assay

1:20000

Immunocytochemistry/ Immunofluorescence

1:5000

Western Blot

1:10000
Application Notes
The emission spectra for this DyLight(TM) conjugate match the principle output wavelengths of most common fluorescence instrumentation. This product is designed for immunofluorescence microscopy, fluorescence based plate assays (FLISA) and fluorescent western blotting. This product is also suitable for multiplex analysis, including multicolor imaging, utilizing various commercial platforms.

Formulation, Preparation, and Storage

Purification

Multi-step

Reconstitution

Reconstitute with 100 ul deionized water (or equivalent).

Formulation

Lyophilized from 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 10 mg/mL Bovine Serum Albumin (BSA) - Immunoglobulin and Protease free

Format

Pre-adsorbed

Preservative

0.01% Sodium Azide

Concentration

LYOPH mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store lyophilized antibody at 4C in the dark. Aliquot reconstituted liquid and store at -20C. Avoid freeze-thaw cycles.

Background: IgG (H+L)

Antibodies, also known as immunoglobulins (Igs) are critical for immunity and are grouped into five primary classes: IgG, IgM, IgA, IgD, and IgE. The most abundant antibody isotype is immunoglobulin G (IgG) with concentrations ranging from 7.5-22 mg/ml in human serum and has a molecular weight of 150 kDa. The major effector functions of IgG include neutralization, opsonization, complement fixation and antibody dependent cell-mediated cytotoxicity (ADCC). This monomeric immunoglobulin, expressed on the surface of mature B cells, is often depicted as a Y-shape and comprised of 2 heavy chains and 2 light chains linked by disulfide bonds. The heavy chain is type gamma including subtypes gamma 1, gamma 2, gamma 3, and gamma 4 while the light chain is either a kappa or lambda chain. An IgG molecule has two antigen binding sites, each consisting of a heavy and light chain N-terminal variable domain. When combined with the constant heavy chain 1 (Ch1) and the constant light chain domains, it forms the fragment antigen-binding (Fab) region (2 per antibody). The remaining domains (Ch2-Ch4) of both heavy chains make up the Fc region and contain a site for covalently linking an enzymatic or fluorochrome probe, such as HRP or Janelia Fluor 549, for target detection and visualization (1,2,3).

The 4 IgG subclasses, sharing 95% amino acid identity, include IgG1, IgG2, IgG3, and IgG4 for humans and IgG1, IgG2a, IgG2b, and IgG3 for mice. The relative abundance of each human subclass is 60% for IgG1, 32% for IgG2, 4% for IgG3, and 4% for IgG4. In an IgG deficiency, there may be a shortage of one or more subclasses (4).

References

1. Painter RH. (1998) Encyclopedia of Immunology (Second Edition). Elsevier. 1208-1211

2. Chapter 9 - Antibodies. (2012) Immunology for Pharmacy. Mosby 70-78

3. Schroeder H, Cavacini, L. (2010) Structure and Function of Immunoglobulins. J Allergy Clin Immunol. 125(2 0 2): S41-S52. PMID: 20176268

4. Vidarsson G, Dekkers G, Rispens T. (2014) IgG subclasses and allotypes: from structure to effector functions. Front Immunol. 5:520. PMID: 25368619

Additional IgG (H+L) Products

Product Documents

Product Datasheets

Certificate of Analysis

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Product Specific Notices

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Secondary Antibodies are guaranteed for 1 year from date of receipt.

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